Modulation of MRP-1-mediated multidrug resistance by indomethacin analogues

Article abstract of DOI:10.1021/jm0499099

Multidrug resistance (MDR) is a major limiting factor in the development and application of drug candidates. MDR caused by MRP-1 is known to be modulated by the nonsteroidal antiinflammatory drug indomethacin. We have synthesized and biologically evaluated a library of indomethacin analogues. The indomethacin-derived compound library was synthesized employing the Fischer-indole synthesis as the key transformation and making use of a resin-capture-release strategy. Sixty representative members of the library were evaluated in a cell biological cytotoxicity assay employing the MRP-1 expressing human glioblastoma cell line T98G as a model system. Nine of the 60 tested derivatives increased the doxorubicin-mediated cytotoxicity at a comparable or higher level than indomethacin itself. Analysis of these derivatives revealed an interesting structure-function relationship. Most remarkably, two substances increased the toxicity, when doxorubicin was used at clinically relevant low concentrations, at a higher degree than indomethacin.

Full text of DOI:10.1021/jm0499099

J. Med. Chem. 2005, 48, 1179-1187
1179
Modulation of MRP-1-Mediated Multidrug Resistance by Indomethacin
Analogues
Claudia Rosenbaum,† Sonja Ro¨hrs,‡ Oliver Mu¨ller,‡,§ and Herbert Waldmann*,†
Abteilung Chemische Biologie and Abteilung Strukturelle Biologie, Max-Planck-Institut fu¨r Molekulare Physiologie,
Otto-Hahn-Strasse 11, 44227 Dortmund, Germany, and Organische Chemie, Universita¨t Dortmund, Fachbereich 3,
44227 Dortmund, Germany
Received February 2, 2004
Multidrug resistance (MDR) is a major limiting factor in the development and application of
drug candidates. MDR caused by MRP-1 is known to be modulated by the nonsteroidal
antiinflammatory drug indomethacin. We have synthesized and biologically evaluated a library
of indomethacin analogues. The indomethacin-derived compound library was synthesized
employing the Fischer-indole synthesis as the key transformation and making use of a “resin-
capture-release” strategy. Sixty representative members of the library were evaluated in a
cell biological cytotoxicity assay employing the MRP-1 expressing human glioblastoma cell line
T98G as a model system. Nine of the 60 tested derivatives increased the doxorubicin-mediated
cytotoxicity at a comparable or higher level than indomethacin itself. Analysis of these
derivatives revealed an interesting structure-function relationship. Most remarkably, two
substances increased the toxicity, when doxorubicin was used at clinically relevant low
concentrations, at a higher degree than indomethacin.
Introduction
major MDR proteins are highly promiscuous transport-
ers; they share the ability of recognizing and translo-
cating a large number of various, mainly hydrophobic
compounds. In addition to their overlapping substrate
specificity, each transporter can handle unique com-
pounds. P-170 is a transporter for large hydrophobic
either uncharged or slightly positively charged com-
pounds, while the MRP family is mostly transporting
hydrophobic anionic conjugates and also uncharged
drugs. MRP mediates also the transport of partially
detoxified compounds, such as glutathione and glucu-
ronide conjugates.6 Compounds such as verapamil or
cyclosporine can circumvent P-170-mediated MDR. How-
ever, clinical studies failed to meet expectations because
the use of these drugs in a combined chemotherapy
caused severe negative side effects.7 Until today there
are few data about MDR caused by MRP-1 and its
circumvention.8 Thus, the development of potent and
nontoxic agents, which unfold minimal side effects, to
inhibit MRP-1-induced MDR belongs to the crucial aims
of medical and pharmacological research. Recently
nonsteroidal antiinflammatory drugs (NSAIDs) turned
into the focus of medical interest because of their high
potential as agents in cancer prevention.9 Several stud-
ies analyzed the NSAID-induced increase of chemo-
therapeutical toxicity.10 These studies show that MRP-1
activity can be modulated by treatment with the NSAID
indomethacin (1).11 One possible explanation for this
modulation is a direct interaction between indomethacin
and MRP-1 and the competitive inhibition of MRP-1.
This model is supported by the fact that indomethacin
with a pKa ) 4.1 is negatively charged at physiological
pH and that MRP-1 shows a preference for hydrophilic
compounds and transports amphiphilic organic an-
ions.12,5 Studies with indomethacin have also shown
that MDR is probably not caused by the inhibition of
glutathione-S-transferase (GST). Compounds, which are
A large number of clinically observed resistance of
cancers to chemically unrelated cytotoxic compounds is
caused by the overexpression of multidrug transporter
proteins in the tumor cell membrane. Multidrug resis-
tance (MDR) describes the property of a cell to develop
resistance against the effects of a certain external
compound and in some cases also against structurally
and functionally different compounds. Many heterocyclic
natural compounds, which are used in tumor therapy,
can cause MDR. Among these are the alkaloids dauno-
rubicine, doxorubicin, and vincristine. MDR can result
in a significant efficiency decrease of chemotherapy.
Many studies have analyzed the development of MDR
in different cell types. The best characterized mecha-
nism of MDR is the overexpression of the gene MDR-1,
which codes for the transmembrane protein phospho-
glycoprotein P-170.1 This protein works as an ATP-
dependent pump and is able to lower the intracellular
concentration of several cytotoxic compounds. P-170
shows a sequence similarity of 15-19% to the family of
multidrug resistance associated proteins (MRPs), which
consists of at least seven homologous members.2 MRPs
are able to cause MDR, which is independent from
P-170.3 The 190 kDa MRP-1 is found in normal human
and tumor tissues. On the basis of their sequence
homologies P-170 and the MRP family belong to the
class of transmembrane ABC transporters.4 P-170 and
MRP-1 have similar mechanisms of drug export.5 The
* To whom correspondence should be addressed. Phone: +49-231-
133-2400. Fax: +49-231-133-2499. E-mail: herbert.waldmann@
mpi-dortmund.mpg.de.
† Abteilung Chemische Biologie, Max-Planck-Institut fu¨r Molekulare
Physiologie, and Universita¨t Dortmund.
‡ Abteilung Stukturelle Biologie, Max-Planck-Institut fu¨r Moleku-
lare Physiologie.
§ Phone: +49-231-133-2158. Fax: +49-231-133-2199. E-mail:
oliver.mueller@mpi-dortmund.mpg.de.
10.1021/jm0499099 CCC: $30.25 © 2005 American Chemical Society
Published on Web 01/25/2005

1180 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4
Rosenbaum et al.
Figure 1. Three-step synthesis of the indomethacin library by a resin “capture-and-release” methodology.
unable to inhibit GST, can still inhibit cellular detoxi-
fication to a level similar to that of indomethacin.10
Correlations between MDR inhibition and the well-
known inhibitory effects on the cyclooxygenase (COX)
pathway by indomethacin could be excluded.13,14 Thus,
the two isoforms of the COX enzyme COX-1 and COX-2
seem to play no relevant role in the mechanism of MDR
inhibition. On the basis of the current data, indometha-
cin might serve as a promising amplifier of chemothera-
peutical toxicity and might be suited for the combined
cytostatic therapy of cancer types, which have developed
MDR as a result of MRP-1 overexpression. In this study
we analyzed the MDR modulation induced by novel
indomethacin analogues in order to identify compounds
which show stronger effects on MDR compared to
indomethacin. To this end, we synthesized a library of
indomethacin analogues in a solid-phase synthesis
approach and characterized the compounds in a com-
bined toxicity assay.
Chemtech) was condensed with hydrazines 3 to yield
polymer-bound hydrazones 4. In intermediates 4 the
polymer serves as reagent-capturing auxiliary, allowing
for easy removal of surplus reagent and as temporary
blocking function for the terminal NH2 group of the
hydrazine. Regioselectively N-acylated hydrazones 6
obtained thereby are then subjected to treatment with
acid and ketones 8 in the presence of water traces to
yield the desired indole derivatives 10. In this reaction
sequence the water hydrolyzes the hydrazones and
thereby releases selectively acylated hydrazines 7 into
solution where they condense with ketones 8 to give
hydrazone intermediates 9. These then undergo a [3,3]-
sigmatropic rearrangement under the reaction condi-
tions, thereby shifting the equilibrium between the
hydrazones 6 and 9 irreversibly to the desired side. This
strategy employs ketones, acid chlorides, and hydrazines
as building blocks which are either commercially avail-
able in a great variety or readily prepared by numerous
well-established synthesis methods allowing for ready
construction of a fairly diverse compound collection.
Figure 2 shows the building blocks used for the
synthesis of a library of 197 indomethacin analogues
(see the Supporting Information) in overall yields rang-
ing from 4% to quantitative (see Table 1).
The results demonstrate that electron-withdrawing
and -donating substituents can be incorporated in the
hydrazine and the acid chloride. Electron-poor and -rich
heterocycles are tolerated as well. Also the ketones may
incorporate different heteratom substituents. The use
of hydrazine building blocks (A-H) and substituted
benzoyl chlorides (I-IX) lead to formation of aromatic
indoles, which carry the substituents in positions 5 and
7 and a N-benzoyl unit with different para-substituents
reaching from sterically demanding and nonpolar bi-
phenyl rings IV to tert-amines IX. The diversity of the
used ketones (1-16) ranges from the hydrophilic lae-
vulinic acid 9 as part of indomethacin to the very
hydrophobic 2-tridecanone 11. Not unexpectedly the
overall yield was highest if activating electron-donating
Results and Discussion
Chemistry. The indomethacin-derived compound
library was synthesized by employing the Fischer-
indole synthesis as a key step.15 To establish a particu-
larly practical and efficient synthesis sequence, we
resorted to a “resin-capture-release” strategy16 that
made use of three types of readily available building
blocks and circumvented permanent attachment to and
final release from the solid support in additional syn-
thesis steps.17
As shown in Figure 1 polystyrene aldehyde resin 2
(loading, 0.9 mmol‚g-1
; obtained from Advanced

Multidrug Resistance by Indomethacin Analogues
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4 1181
Figure 2. Scheme of the library building blocks.
Table 1. Results of the Library Synthesis
yielda (%)
yielda (%)
A-I
45-100
9-26
C-IX
D-I
20-47
47
A-V
A-VII
B-I
C-I
C-II
C-III
C-IV
C-VI
C-VII
C-VII I
62-100
21-60
19-45
16-63
32-81
15-81
17-49
9-99
E-I
40-90
4-58
4-52
15-20
7-55
11-66
10-29
4-26
11-29
F-I
F-IV
F-VI
F-VII
F-VII I
F-IX
G-I
21-46
H-I
a Yield ranges are given for the conversion of the respective
N-acylated hydrazones with ketones (1-16). All yields refer to
purified products (purity > 99% (HPLC, 215 nm)). The identities
of the compounds were proven by means of LC-MS, GC-MS, NMR,
and high-resolution mass spectra.
substituents were incorporated into the hydrazines,
indicating the importance of two hydrazone formation
reactions in the overall process. The use of the electroni-
cally activated phenylhydrazine blocks C and E resulted
in very high yields. However, even in the presence of
deactivating SO3H, COOH, and NO2 groups, the syn-
thesis of N-acylated indoles via the “resin-capture-
release” strategy was successful.
Depending on the ketone employed, the crude reaction
products are obtained with purities of approximately 70
to >95%. After simple chromatography, all compounds
were isolated in >99% purity.
Figure 3. Analysis of MRP expression by RT-PCR and of the
presence of the MRP-1 protein in T98G cells. (A) Upper,
agarose gel of MRP transcript after reverse transcription and
PCR amplification; lower, agarose gel of â-actin transcript after
RT-PCR: M, 100 bp ladder marker; T98G, RNA from 98G cells;
C, template-free negative control. The sizes of marker frag-
ments are indicated in base pairs. (B) Western blot membrane
probed with anti-MRP-1 antibody: SN, soluble supernatant;
TL, total cell lysate of T98G cells. The sizes of molecular weight
marker fragments are indicated in kilodaltons.
These synthesis strategies gave rise to a complex and
diverse indole library synthesized efficiently within a
very short period of time. For the cell-based assay we
first selected several representative compounds for each
building block and combined them, in a positional
scanning mode, with the indomethacin core structure.
We focused on the active substituents and selected
compounds for the next cell assay based on these
results. Over all 60 indomethacin analogues were evalu-
ated in a combined toxicity assay (Table 2).

1182 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4
Table 2. List of Indomethacin Analogues Investigated in This Study
Rosenbaum et al.
compd
R1
R2
R3/R4
name
1
C
C
C
C
C
C
C
C
C
A
C
C
C
C
C
F
F
F
F
F
F
F
F
F
F
F
E
E
F
F
F
F
F
F
F
F
F
F
C
C
G
G
G
G
F
F
F
F
F
F
F
F
F
F
C
C
C
C
C
C
I
9
[1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl]acetic acid
[1-(4-fluorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl]acetic acid
[1-(4-bromobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl]acetic acid
[1-(biphenyl-4-carbonyl)-5-methoxy-2-methyl-1H-indol-3-yl]acetic acid
[5-methoxy-2-methyl-1-(4-methylbenzoyl)-1H-indol-3-yl]acetic acid
[5-methoxy-2-methyl-1-(pyridine-2-carbonyl)-1H-indol-3-yl]acetic acid
1-[1-(furan-2-carbonyl)-5-methoxy-1H-indol-2-yl]ethanone
[1-(furan-2-carbonyl)-5-methoxy-2-methyl-1H-indol-3-yl]acetic acid
[5-methoxy-2-methyl-1-(thiophene-2-carbonyl)-1H-indol-3-yl]acetic acid
[5-bromo-1-(furan-2-carbonyl)-2-methyl-1H-indol-3-yl]acetic acid
3-[1-(4-bromobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl]propionic acid
4-[1-(4-bromobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl]butyric acid
2
II
9
3
III
IV
V
9
4
9
5
9
6
VI
VII
VII
VIII
VII
III
III
IV
VI
VI
I
9
7
3
8
9
9
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
9
10
14
10
10
14
1
3-[1-(biphenyl-4-carbonyl)-5-methoxy-2-methyl-1H-indol-3-yl]propionic acid
3-[5-methoxy-2-methyl-1-(pyridine-2-carbonyl)-1H-indol-3-yl]propionic acid
4-[5-methoxy-2-methyl-1-(pyridine-2-carbonyl)-1H-indol-3-yl]butyric acid
9-(4-chlorobenzoyl)-6,7,8,9-tetrahydro-5H-carbazole-3-sulfonic acid
I
4
1-(4-chlorobenzoyl)-3-ethyl-2-methyl-1H-indole-5-sulfonic acid
I
5
5-(4-chlorobenzoyl)-2-methyl-2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indole-8-sulfonic acid
9-(4-chlorobenzoyl)-1,2,4,9-tetrahydro-3-thia-9-aza-fluorene-6-sulfonic acid
5-(4-chlorobenzoyl)-6,7,8,9,10,11-hexahydro-5H-cycloocta[b]indole-2-sulfonic acid
1-(4-chlorobenzoyl)-3-decyl-2-methyl-1H-indole-5-sulfonic acid
I
6
I
7
I
11
12
14
14Me
15
16
10
14
1
I
3-benzyl-1-(4-chlorobenzoyl)-2-methyl-1H-indole-5-sulfonic
4-[1-(4-chlorobenzoyl)-2-methyl-5-sulfo-1H-indol-3-yl]butyric acid
4-[1-(4-chlorobenzoyl)-2-methyl-5-sulfo-1H-indol-3-yl]butyric acid methyl ester
2-benzoyl-5-(4-chlorobenzoyl)-2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indole-8-sulfonic acid
1-(4-chlorobenzoyl)-3-(4-hydroxybenzyl)-2-methyl-1H-indole-5-sulfonic acid
3-[1-(4-chlorobenzoyl)-2,5-dimethyl-1H-indol-3-yl]propionic acid
4-[1-(4-chlorobenzoyl)-2,5-dimethyl-1H-indol-3-yl]butyric acid
9-(furan-2-carbonyl)-6,7,8,9-tetrahydro-5H-carbazole-3-sulfonic acid
3-ethyl-1-(furan-2-carbonyl)-2-methyl-1H-indole-5-sulfonic acid
9-(furan-2-carbonyl)-1,2,4,9-tetrahydro-3-thia-9-azafluorene-6-sulfonic acid
3-[1-(furan-2-carbonyl)-2-methyl-5-sulfo-1H-indol-3-yl]propionic acid
3-[1-(furan-2-carbonyl)-2-methyl-5-sulfo-1H-indol-3-yl]propionic acid methyl ester
3-decyl-1-(furan-2-carbonyl)-2-methyl-1H-indole-5-sulfonic acid
I
I
I
I
I
I
VII
VII
VII
VII
VII
VII
VII
VII
VII
VII
I
4
6
10
10Me
11
12
14
14Me
15
10
14
1
3-benzyl-1-(furan-2-carbonyl)-2-methyl-1H-indole-5-sulfonic acid
4-[1-(furan-2-carbonyl)-2-methyl-5-sulfo-1H-indol-3-yl]butyric acid
4-[1-(furan-2-carbonyl)-2-methyl-5-sulfo-1H-indol-3-yl]butyric acid methyl ester
2-benzoyl-5-(furan-2-carbonyl)-2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indole-8-sulfonic acid
3-[1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl]propionic acid
4-[1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl]butyric acid
9-(4-chlorobenzoyl)-6,7,8,9-tetrahydro-5H-carbazole-3-carboxylic acid
9-(4-chlorobenzoyl)-1,2,4,9-tetrahydro-3-thia-9-azafluorene-6-carboxylic acid
1-(4-chlorobenzoyl)-3-decyl-2-methyl-1H-indole-5-carboxylic acid
3-(3-carboxypropyl)-1-(4-chlorobenzoyl)-2-methyl-1H-indole-5-carboxylic acid
9-(biphenyl-4-carbonyl)-6,7,8,9-tetrahydro-5H-carbazole-3-sulfonic acid
5-(biphenyl-4-carbonyl)-2-methyl-2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indole-8-sulfonic acid
9-(biphenyl-4-carbonyl)-1,2,4,9-tetrahydro-3-thia-9-azafluorene-6-sulfonic acid
1-(biphenyl-4-carbonyl)-3-decyl-2-methyl-1H-indole-5-sulfonic acid
3-benzyl-1-(biphenyl-4-carbonyl)-2-methyl-1H-indole-5-sulfonic acid
4-[1-(biphenyl-4-carbonyl)-2-methyl-5-sulfo-1H-indol-3-yl]butyric acid
2-benzoyl-5-(biphenyl-4-carbonyl)-2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indole-8-sulfonic acid
1-(biphenyl-4-carbonyl)-3-(4-hydroxybenzyl)-2-methyl-1H-indole-5-sulfonic acid
9-(pyridine-2-carbonyl)-6,7,8,9-tetrahydro-5H-carbazole-3-sulfonic acid
9-(pyridine-2-carbonyl)-1,2,4,9-tetrahydro-3-thia-9-azafluorene-6-sulfonic acid
3-[5-methoxy-2-methyl-1-(thiophene-2-carbonyl)-1H-indol-3-yl]propionic acid ethyl ester
4-[5-methoxy-2-methyl-1-(thiophene-2-carbonyl)-1H-indol-3-yl]butyric acid
[1-(4-(dimethylamino)benzoyl)-5-methoxy-2-methyl-1H-indol-3-yl]acetic acid
3-[1-(4-(dimethylamino)benzoyl)-5-methoxy-2-methyl-1H-indol-3-yl]propionic acid
4-[1-(4-(dimethylamino)benzoyl)-5-methoxy-2-methyl-1H-indol-3-yl]butyric acid
4-[1-(4-(dimethylamino)benzoyl)-5-methoxy-2-methyl-1H-indol-3-yl]butyric acid ethyl ester
I
I
I
6
I
11
14
1
I
IV
IV
IV
IV
IV
IV
IV
IV
VI
VI
VIII
VIII
IX
IX
IX
IX
5
6
11
12
14
15
16
1
6
10Et
14
9
10
14
14Et
Evaluation of Cytotoxicity. The human glioblas-
toma cell line T98G, which is known to express MRP-
1,14 was chosen for the biological assays. MRP expres-
sion was analyzed by reverse transcription and PCR.
The MRP transcript of the expected size was detected
in T98G cells of the same passaging step as that used
in the further assays (Figure 3A).14 Next we confirmed
the translation of the MRP transcript. The clear band
at the approximate size of the MRP-1 protein in the
Western blot showed that the protein is present in T98G
cells (Figure 3B). Not surprisingly, the relative amount
of the membrane protein MRP-1 was higher in the total
lysate, which comprises the membrane fraction, as
compared to the soluble fraction of the cells. Into a 96-
well plate, 104 cells/well were seeded. During the initial
phase of assay optimization, concentrations of indometha-
cin analogues were between 1.25 and 160 µM and
doxorubicin concentrations were between 0.08 and 40
µM. In the final assay indomethacin analogues were
used at 5-10 µM and doxorubicin at 0.1-1.0 µM. The
final dimethyl sulfoxide (DMSO) concentration over a
period of 4 days did not exceed 0.5%. As tested in a
control experiment, this DMSO concentration is not
toxic for T98G cells (not shown). Figure 4 shows the cell

Multidrug Resistance by Indomethacin Analogues
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4 1183
Figure 4. Indomethacin sensitizes T98G human malignant glioma cells to doxorubicin cytotoxicity. T98G cells were pretreated
with indomethacin [c(indomethacin) ) 1-50 µM] for 2 h and then cotreated with doxorubicin [c(doxorubicin) ) 0-10 µM] for (A)
3 or (B) 4 days. Survival was measured by crystal violet staining as described in Materials and Methods. Data are expressed as
the mean percentage of survival (SD < 10%; n ) 3).
survival rate at six different indomethacin concentra-
tions in dependence of the doxorubicin concentration
after 3 and 4 days. The results show that the signal-
to-noise ratio is significant after 4 days. Thus, the
following assays to measure the combined toxicity of the
potential MRP-1 inhibitors and the cytostatic drug
doxorubicin were performed after an incubation time
of 4 days.
In a preliminary experiment we tested the potentially
inhibiting effects of 60 different indoles at a concentra-
tion of 10 µM without or with 0.2, 0.5, or 1 µM
doxorubicin. The results showed the influence of the
indole compounds on the cytotoxicity of doxorubicin
(Figure 5). On the basis of these results, we classified
the derivatives into three groups (Table 3). Group a
comprises 24 molecules, which neither increased the
cytotoxic effects of doxorubicin nor had any other effect
on cell survival under the applied conditions. In group
b the substances were classified, which increased doxo-
rubicin induced cell mortality. The effect of these 23
compounds was at least 10% weaker than the effect
shown with indomethacin. Group c comprises nine
molecules, which increased the cytotoxicity of doxoru-
bicin at the same or at an even 13% higher level than
indomethacin itself. Finally category d comprises toxic
substances, which caused an increased general cell
mortality at 10 µM even in the absence of doxorubicin.
The incubation with the four agents of this group
decreased the cell number by at least 30% compared to
the untreated control. Such toxic effects circumvent the
differentiation between the toxicity of the compound and
a potential inhibition of MRP-1. Thus, the compounds
of group d could not be evaluated in the assay.
The classification of all 60 compounds according to
these criteria allowed first important conclusions about
the structure-function relationships (Table 3). The
replacement of the methoxy moiety at the hydrazine
building block C (Figure 2) by bromine (A) or by sulfonic
acid (F) leads to the loss of activity. Also fluoryl and
nicotinic acid chloride building blocks (VII and X) led
to inactive indoles. All tested esters showed lower
activities compared to the free acid and often showed
an increased toxicity. One possible explanation for lower
activity of the esters could be a direct interaction

1184 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4
Rosenbaum et al.
Figure 5. Exemplified results of preliminary experiments performed at a constant concentration to identify potentially interesting
compounds. T98G cells were pretreated with indomethacin [c(indomethacin) ) 10 µM] for 2 h and then cotreated with doxorubicin
[c(doxorubicin) ) 0-1.0 µM] for 4 days. Survival was measured by crystal violet staining as described in Materials and Methods.
Data shown are the mean percentage ( SD for a minimum of three experiments.
Table 3. Summary of the Cytotoxicity Assay Results and Classification of All Nonsteroidal Antiinflammatory Compounds According
Their Effects at c(compound) ) 10 µM
classification
compound no.
(a) inactivea
6, 8, 10, 14, 16, 17, 19, 20, 21, 22, 24, 29, 30, 31, 32, 33, 34, 36, 37, 38, 46, 49, 50, 52
1, 2, 4, 5, 7, 9, 13, 15, 25, 26, 43, 44, 45, 47, 51, 53, 54, 56, 57, 58, 59, 60
3, 11, 12, 27, 28, 39, 40, 41, 42
(b) activeb
(c) very activec
(d) toxicd
18, 23, 35, 48, 55
a Inactive compounds had no synergistic effect on the doxorubicin-induced cell death at c(doxorubicin) ) 0.1 µM. b Active compounds
increased doxorubicin-induced cell mortality at least 10% lower than indomethacin at c(doxorubicin) ) 0.1 µM. a Very active compounds
increased doxorubicin-induced cell mortality to a similar or up to 13% higher extent than indomethacin at c(doxorubicin) ) 0.1 µM. The
general structures of these compounds are given in Figure 8. The detailed assay results of all compounds of category c are given in the
Supporting Information. d Toxic compounds increased general cell mortality even in the absence of doxorubicin. At the NSAID concentrations
of 10 µM the cell survival was <70%. Therefore these compounds were not evaluated any further.
between the carboxylic acid and MRP-1. This model is
supported by the fact that MRP-1 shows a preference
for hydrophilic compounds and transports amphiphilic
organic anions.12, 5
The nine active compounds categorized in group c
(Figure 6) were analyzed in further detail. These
analyses were performed with 5 µM indole concentration
and 0.1, 0.2, 0.5, and 0.8 µM doxorubicin concentration.
All measurements were done in triplicate. In these
experiments the seven compounds 3, 27, 28, 39, 40, 41,
and 42 induced a high increase in doxorubicin toxicity.
All these compounds showed an inhibition of MRP-1 at
a level similar to that of indomethacin. Remarkably,
derivatives 11 and 12 showed an even higher inhibition
compared to indomethacin (Table 4). On the basis of the
data shown in Table 4, the cell mortalities induced by
indomethacin and analogues 11 and 12 in a combined
application with doxorubicin were correlated to the cell
mortality of doxorubicin alone. Figure 7 illustrates, e.g.,
that indomehtacin (at 5µM) in combination with doxo-
Figure 6. Structures of the nine active indomethacin ana-
rubicin (at 0.1 µM) increases the cell death in the
logues (category c, Table 3).
described assay conditions compared to doxorubicin
alone up to 9% and the cell mortality is amplified to
22% when compound 11 is used in the same assay
system. This 2.4-fold increased cytotoxicity induced by
indomethacin analogues 11 demonstrates the potential
of the combinatorial chemistry approach with the in-
domethacin core structure. Due to the reason that in
some cancer cell lines the IC50 of doxorubicin is below
0.1 µM, the synergistic effect of indomethacin especially
in this low concentration range is of particular impor-

Multidrug Resistance by Indomethacin Analogues
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4 1185
Table 4. Demonstration of a Synergistic Combination of
Selected Nonsteriodal Antiinflammatory Drugs (NSAIDs) with
Doxorubicin as the Chemotherapeutic Drug in T98G Cellsa
Experimental Section
General Methods for Chemical Synthesis. Materials.
Unless otherwise noted, reagents were purchased from Acros
Chimica, Fluka, Sigma, and Aldrich and used without further
purification. LC-MS was performed on the 1100 series from
Hewlett-Packard with a VP 50/10 Nucleosil C18PPN column
(Macherey-Nagel) and a Finnigan LCQ ESI spectrometer with
a gradient: 90/10 (v/v) H2O/acetonitrile (0.1% trifluoroacetic
acid) to 10/90 (v/v) in 30 min; flow, 1 mL/min. Preparative
HPLC was conducted by using Pro Star 215/Varian HPLC with
a VP 250/21 Nucleosil C18PPN column (Macherey-Nagel) and
a gradient: 90/10 (v/v) H2O/acetonitrile (0.1% trifluoroacetic
acid) to 10/90 (v/v) in 30 min; flow, 20 mL/min. Nuclear
magnetic resonance (NMR) spectra were recorded on a Varian
Mercury 400 (400 MHz 1H NMR; 100.6 MHz 13C NMR). 1H
NMR spectra are tabulated in the following order: chemical
shifts calculated with reference to solvent standards based on
tetramethylsilane, multiplicity (s, singulet; d, doublet; t, triplet;
q, quartet; m, multiplet), coupling constants in Hz, and number
of protons. The 70 eV electron ionization (EI) high-resolution
mass spectra (HR-MS) were recorded on Finnigan MAT MS
70 spectrometer.
General Procedure for Hydrazone Formation. The
aldehyde resin (0.5 g, 0.55 mmol) was dried in high vacuum
overnight and suspended in 5 mL of dichloroethane (DCE).
To this suspension, 2.75 mmol (5 equiv) of hydrazine hydro-
chloride and 194 µL (6 equiv) of triethylamine (NEt3) were
added under argon atmosphere. The mixture was shaken at
45 °C overnight. After cooling the resin was filtered and
washed three times with each 5 mL of N,N-dimethylformamide
(DMF), 90/10 (v/v) DMF/H2O, DMF, dichloromethane, ethyl
acetate, and methanol.
no
NSAID
indomethacin
11
(5 µM)
12
(5 µM)
(5 µM)
no DOX
100.0 ( 4.3 100.9 ( 1.3 95.5 ( 3.3 95.2 ( 2.1
0.1 µM DOX 75.8 ( 4.9
0.2 µM DOX 71.4 ( 2.2
0.5 µM DOX 37.2 ( 1.0
69.3 ( 0.3 59.0 ( 2.6 63.6 ( 2.3
56.6 ( 3.2 48.6 ( 1.9 53.7 ( 3.9
18.7 ( 2.1 11.6 ( 2.6 16.9 ( 2.3
a Data are expressed as percentage cell survival ( standard
deviation for a minimum of three determinations. The NSAID
concentrations were nontoxic (cell survival > 95%) in all cases.
tance.10 Especially at this clinically relevant low doxo-
rubicin concentration of 0.1 µM the two novel com-
pounds showed significant effects and no general toxicity
in the absence of doxorubicin.
The goal of our investigation was to identify novel
compounds which are more potent inhibitors of MDR
observed in the presence of the cytostatic therapeutic
doxorubicin. Overall we analyzed a library of 60 in-
domethacin analogues in a combined toxicity cell bio-
logical assay. The results revealed nine very active
compounds. Compound 11 displayed an increase of the
doxorubicin-induced cytoxicity by a factor of 2.4 and
compound 12 by a factor of 1.7. Thus, structure 11
represents an attractive starting point for the develop-
ment of potent MRP-1 inhibitors.
All compounds classified as very active showed a high
structural homology to indomethacin (Figure 8). The
extension of the alkyl chain by a methylene group in
11 led to an activity increase in comparison to in-
domethacin. However, the additional CH2 group in 12
has no further effect. Remarkably, only the use of
4-halogen-substituted benzoyl building blocks led to
active compounds. The most active compounds 11 and
12 carry a bromine substituent. As molecules 27 and
28 demonstrate, the methoxy group of indomethacin can
also be substituted by a methyl group without complete
loss of activity. On first glance the high activities of 41
and 42 seem surprising, because these molecules do not
have the 3-indole acetic acid motif of indomethacin.
However, closer inspection of the structure shows that
the acid function of 41 is in comparable distance to the
central pyrrole as in 11 (see Figure 9). The free car-
boxylic acid in 41 is bound to the aromatic six-membered
ring of the indole, which carries a nonsubstituted
nonpolar ring as an equivalent to the methoxyphenyl
motif in 11. This structural relationship indicates a rigid
donor acceptor relation between the free acid and the
N-benzoyl motif and amino acids of MRP-1, which may
be crucial for MRP-1 activity.
In the present report we have described a synthesis
procedure and SAR studies based on the known MRP-1
inhibitor 1. As a result, two novel and potent MRP-1
modulators with no cytotoxcitiy (cell survival > 95% at
cmodulator < 10 µM) were identified. We have demon-
strated that these indomethacin analogues have the
ability to potentiate the toxicity of the chemotherapeutic
agent doxorubicin, and they are an attractive starting
point for further development of MRP-1 inhibitors. In
some cancers, where drug resistance is a result of
MRP-1 overexpression, this synergistic effect of NSAIDs
with chemotherapeutic agents potentially improves
existing treatments for cancer.
General Procedure for Hydrazone Acylation. The
hydrazone resin (0.5 g, 0.45 mmol) was dried in high vacuum
overnight and suspended in 5 mL of pyridine. To the mixture
1.35 mmol (3 equiv) of acid chloride was added under argon.
The mixture was shaken at 80 °C overnight. After cooling the
resin was filtered and washed three times with N,N-dimeth-
ylformamide (DMF), 90/10 (v/v) DMF/H2O, DMF, dichlo-
romethane, ethyl acetate, and methanol.
General Procedure for Cleavage and Indole Rear-
rangement. The acylated hydrazone resin (150 mg, 0.11
mmol) was suspended in 6 mL of DCE/TFA (1/1). The corre-
sponding ketone (10 equiv) was added, and the mixture was
heated for 15 min to 2 h at 70 °C. After cooling the mixture
was quenched with methanol and the resin was filtered and
washed with 5 mL of dichloromethane, methanol, ethyl
acetate, and methanol. The filtrate was evaporated to dryness,
and the crude product was purified by preparative HPLC.
Analytical Data for Selected Compounds. (a) [1-(4-
Bromobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl]ace-
tic Acid (3; C-III-9). White solid; yield 57%; HPLC purity >
99% (215 nm); mp 151 °C. 1H NMR (400 MHz, CD3OD): δ 7.73
(d, 3J ) 8.2 Hz, 2 H, arom CH), 7.60 (d, 3J ) 8.2 Hz, 2 H,
4
3
arom CH), 7,00 (d, J ) 2.5 Hz, 1 H, arom CH), 6.91 (d, J )
3
4
8.6 Hz, 1 H, arom CH), 6.67 (dd, J ) 8.6 Hz, J ) 2.5 Hz, 1
H, arom CH), 3.80 (s, 3 H, OCH3), 3.69 (s, 2 H, CH2), 2.31 (s,
3 H, CH3). MS (ESI): 400.0 [M - H]- . HR-MS (FAB, m/z).
Calcd for C19H15BrNO4 [M - H]-: 400.0185. Found: 400.0181.
(b) 3-[1-(4-Bromobenzoyl)-5-methoxy-2-methyl-1H-in-
dol-3-yl]propionic Acid (11; C-III-10). White solid; yield
25%; HPLC purity > 99% (215 nm); mp 210 °C. 1H NMR (400
MHz, CD3OD): δ 7.73 (d, 3J ) 8.2 Hz, 2 H, arom CH), 7.60 (d,
3J ) 8.2 Hz, 2 H, arom CH), 7,00 (d, J ) 2.5 Hz, 1 H, arom
4
3
3
CH), 6.91 (d, J ) 8.6 Hz, 1 H, arom CH), 6.67 (dd, J ) 8.6
4
Hz, J ) 2.5 Hz, 1 H, arom CH), 3.80 (s, 3 H, OCH3), 2.81 (t,
3J ) 7.6 Hz, 2 H, CH2), 2.49 (t, 3J ) 7.6 Hz, 2 H, CH2), 2.29 (s,
3 H, CH3). MS (ESI): 414.39; 416.36 [M - H]-. HR-MS (FAB,
m/z). Calcd for C20H17BrNO4 [M - H]-: 414.0341. Found:
414.0346.
(c) 4-[1-(4-Bromobenzoyl)-5-methoxy-2-methyl-1H-in-
dol-3-yl]butyric Acid (12; C-III-14). Green solid; yield 71%;
HPLC purity > 99% (215 nm); mp 210 °C. 1H NMR (400 MHz,
CD3OD): δ 7.70 (d, 3J ) 8.2 Hz, 2 H, arom CH), 7.57 (d, 3J )

1186 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4
Rosenbaum et al.
Figure 7. Demonstration of cell death increase in a combined application of doxorubicin and NSAIDs. The doxorubicin-induced
cell death is correlated to the cell death caused by the addition of NSAIDs. The chart illustrates the activities of the compounds
indomethacin, 11, and 12 in comparison to doxorubicin.
4
3
3J ) 9 Hz, J ) 2,0 Hz, 1 H, arom CH), 7.66 (d, J ) 8.6 Hz,
3
2 H, arom CH), 7.54 (d, J ) 8.6 Hz, 2 H, arom CH), 7.24 (d,
3J ) 9 Hz, 1 H, arom CH), 2.73 (t, J ) 6 Hz, 2 H, CH2), 2.57
3
(t, J ) 6 Hz, 2 H, CH2), 1.82-2.03 (m, 4 H, CH2). 13C NMR
3
(100.6 MHz, CDCl3): δ 210.19 (CdO), 179.37 (CdO), 139.26
(Cq), 137.59 (Cq), 137.29 (Cq), 139.97 (Cq), 131,02 (arom CH),
129.15 (arom CH), 127.35 (Cq), 125.12 (Cq), 124.83 (arom CH),
120.20 (arom CH), 118.28 (Cq), 114.00 (arom CH), 25.77 (CH2),
23.58 (CH2), 22.33 (CH2), 21,05 (CH2). MS (ESI): 352.16 [M
- H]-. HR-MS (FAB, m/z): Calcd for C20H15ClNO3 [M - H]-:
352.0741. Found: 352.0767.
Figure 8. General structures of the nine very active indole
derivatives (category c, Table 3).
(i) 9-(4-Chlorobenzoyl)-1,2,4,9-tetrahydro-3-thia-9-aza-
fluorene-6-carboxylic Acid (42; G-I-11). White solid; yield
6%; HPLC purity > 99% (215 nm). LC-MS: 370.11 [M - H]-.
Rt ) 5.57 min; C19H14ClNO3S, 371.8381 g/mol.
Reverse Transcription PCR. The expression of the MRP
gene was controlled by reverse-transcription PCR (RT-PCR)
of the MRP transcript. RNA was purified from T89G cells by
standard methods (RNeasy Mini Kit, Qiagen). For PCR the
purified RNA was reverse-transcribed into cDNA using Om-
niscript Reverse Transkriptase (Qiagen). The following primers
were used for the PCR of the MRP cDNA:14 MRP sense, 5′-
CGTGTACTCCAACGCTGAC-3′; MRP antisense, 5′-CTGGAC-
CGCTGACGCCCGTGAC-3′. The template was denatured for
5 min at 95 °C, and the PCR was carried out for 35 cycles,
which included 45 s at 95 °C, 45 s at 55 °C, and 60 s at 72 °C.
The MRP product was expected to be of a length of 326 bp.
For positive control, â-actin was directly amplified by OneStep
RT-PCR (Qiagen) from RNA. The used primers were as
follows: â-actin sense 5′-GCGGGATCCTCGACAACGGCTC-
CGGCAT-3′; â-actin antisense 5′-GCGGTCGACGGATCT-
TCATGAGGTAGTCAG-3′. The RT-PCR program was as fol-
lows: 30 min at 50 °C, 15 min at 95 °C, and 35 cycles at 30 s
at 94 °C, 30 s at 60 °C, and 60 s at 72 °C. The resulting â-actin
fragment was expected to be of a length of 628 bp. The PCR
result was controlled by electrophoresis in 2% agarose gels
using a 100 bp ladder marker (Fermentas). Gels were stained
by ethidium bromide.
Western Blot. The T98G cells were analyzed for the
presence of the MRP-1 protein. Subconfluent cells were
harvested and resuspended in PBS including a mix of protease
inhibitors (Complete from Roche). Cells were lysed on ice by
ultrasound sonication. The total lysate was centrifuged for 10
min at 13 000 rpm. The resulting supernatant and the total
lysate were adjusted to equal protein concentrations as
determined by Bradford protein concentration determination.
A 35 mg amount of total protein of each sample was separated
by SDS-PAGE on a 7.5% gel and blotted on a PVDF
membrane (Amersham). The membrane was blocked with
blocking buffer (5% powdered milk, 0.1% Tween20 in PBS) for
2 h. The membrane was incubated with anti-MRP-1 antibody
(QCRL-1 clone from Santa Cruz Biotechnology) at 1:100
dilution in blocking buffer overnight. Next the membrane was
incubated with HRPO-coupled anti-mouse-antibody (Amer-
sham Biosciences) at 1:20 000 dilution in blocking buffer for
1 h at room temperature. The membrane was developed using
Figure 9. Comparative representation of compounds 11 and
41 to demonstrate their structural similarity.
4
8.2 Hz, 2 H, arom CH), 7,08 (d, J ) 2.5 Hz, 1 H, arom CH),
3
6.23 (d, 3J ) 8.6 Hz, 1 H, arom CH), 6.65 (dd, J ) 8.6 Hz, 4J
3
) 2.5 Hz, 1 H, arom CH), 3.81 (s, 3 H, OCH3), 2.73 (t, J )
7,00 Hz, 2 H, CH2), 2.37 (t, 3J ) 7,00 Hz, 2 H, CH2), 2.26 (s, 3
3
H, CH3), 1.91 (q, J ) 7,00 Hz, 2 H, CH2). MS (ESI): 430.23;
428.23 [M - H]-. HR-MS (FAB, m/z). Calcd for C21H19BrNO4
[M - H]-: 428.0497. Found: 428.0505.
(d) 3-[1-(4-Chlorobenzoyl)-2,5-dimethyl-1H-indol-3-yl]-
propionic Acid (27; E-I-10). White solid; yield 23%; HPLC
purity > 99% (215 nm). LC-MS (ESI): 354.64 [M - H]-. Rt )
11.41 min; C20H18ClNO3, 355.8146 g/mol.
(e) 4-[1-(4-Chlorobenzoyl)-2,5-dimethyl-1H-indol-3-yl]-
butyric Acid (28; E-I-14). White solid; yield 25%; HPLC
purity > 99% (215 nm). LC-MS: 368.28 [M - H]-. Rt ) 12,03
min; C21H20ClNO3, 369.84 g/mol.
(f) 3-[1-(4-Chlorobenzoyl)-5-methoxy-2-methyl-1H-in-
dol-3-yl]propionic Acid (39; C-I-10). White solid; yield 39%;
HPLC purity > 99% (215 nm). LC-MS: 370.14 [M - H]-. Rt
) 5.49 min; C21H20ClNO3, 371.8140 g/mol.
(g) 4-[1-(4-Chlorobenzoyl)-5-methoxy-2-methyl-1H-in-
dol-3-yl]butyric Acid (40; C-I-14). White solid; yield 46%;
HPLC purity > 99% (215 nm); mp 105 °C. 1H NMR (400 MHz,
CD3OD): δ 7.64 (d, 3J ) 8.2 Hz, 2 H, arom CH), 7.54 (d, 3J )
4
8.2 Hz, 2 H, arom CH), 7,03 (d, J ) 2.5 Hz, 1 H, arom CH),
3
3
4
6.2 (d, J ) 8.6 Hz, 1 H, arom CH), 6.65 (dd, J ) 8.6 Hz, J
3
) 2.5 Hz, 1 H, arom CH), 3.81 (s, 3 H, OCH3), 2.73 (t, J )
7,00 Hz, 2 H, CH2), 2.37 (t, 3J ) 7,00 Hz, 2 H, CH2), 2.26 (s, 3
3
H, CH3), 1.91 (q, J ) 7,00 Hz, 2 H, CH2). MS (ESI): 384.18
[M - H]-. HR-MS (FAB, m/z). Calcd for C21H19ClNO4 [M -
H]-: 384.1002. Found: 384.1013.
(h) 9-(4-Chlorobenzoyl)-6,7,8,9-tetrahydro-5H-carba-
zole-3-carboxylic Acid (41; G-I-1). White solid; yield 26%;
HPLC purity > 99% (215 nm); mp 254-255 °C. 1H NMR (400
MHz, CDCl3): δ 8.12 (d, 4J ) 2,0 Hz, 1 H, arom CH), 7.78 (dd,

Multidrug Resistance by Indomethacin Analogues
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4 1187
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(17) For a preliminary account of the establishment of this strategy
see: Rosenbaum, C.; Mu¨ller, O.; Baumhof, P.; Mazitschek, R.;
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the ECL Plus Western blot detection system (Amersham
Biosciences).
Cell Line and Cytotoxicity Assay. The human glioblas-
toma cell line T98G was purchased from American Type
Culture Collection (Rockville, MD). Cells were cultured under
standard conditions. Cytotoxicity was evaluated by measuring
the metabolic activity of the cells by using the 3-(4,5-dimeth-
ylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.18
Briefly, the T98G cells were seeded at 104 cells/well in 96-well
plates, adhered for 24 h, and exposed to 10 µM of the drugs
for 4 days. A 100 µL aliquot of the cultured medium was
removed, and the cells were treated with 20 µL of PBS
containing 5 mg/mL MTT (Sigma). After incubation at 37 °C
in a humidified air atmosphere (7.5% CO2) for 2 h, 150 µL of
2-propanol containing 0.04 mol/L HCl were added to each well
to dissolve the formazan crystals produced from the reduction
of MTT by viable cell. After incubation of 45 min and trituation
the absorbance from each well was measured at 620 nm. The
results were expressed as percentages relative to control cells.
The experiments were performed in triplicates and repeated
at least three times.
Acknowledgment. This research was supported by
the Max Planck Gesellschaft and the Fonds der Che-
mischen Industrie. C.R. is grateful to the Landesgra-
duierten Stiftung Baden-Wu¨rttemberg for financial
support. We thank Anette Langerak for excellent tech-
nical assistance.
Supporting Information Available: Biological data con-
cerning the effect of compounds 3, 11, 12, 27, 28, and 39-42
on the modulation of mulitdrug resistance in T98G cells and
analytical data for 23 selected compounds of the indomethacin-
derived library are available. This material is available free
of charge via the Internet at http://pubs.acs.org.
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